The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, gram-positive rods. The genus comprises several species which include, but are not limited to, Mycobacterium africanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. microti, M. scrofulaceum, M. paratuberculosis and M. tuberculosis. Certain of these organisms are the causative agents of disease. For the first time since 1953, cases of mycobacterial infections are increasing in the United States. Although tuberculosis is of particular concern, other mycobacterial infections are also increasing as a result of an increase in the number of immune compromised patients. Many of these new cases are related to the AIDS epidemic, which provides an immune compromised population which is particularly susceptible to infection by Mycobacteria. Mycobacterium avium, Mycobacterium kansasii and other non-tuberculosis mycobacteria are found as opportunistic pathogens in HIV infected and other immune compromised patients.
M. avium and M. intracellulare are members of the Mycobacterium avium complex (MAC). M. paratuberculosis is a subspecies of M. avium and is also generally included in the MAC. These species have become important in recent years because of the high prevalence of disseminated MAC infection in AIDS patients. The Mycobacterium avium complex is comprised of 28 serovars which are distinguishable on the basis of their biochemical and seroagglutination characteristics (see review by Inderlied, et al. 1993. Clin. Microbial. Rev. 6, 266-310). Depending on the method of classification, 10-12 of the 28 serovars are classified as belonging to the species Mycobacterium avium, and 10-12 belong to the species Mycobacterium intracellulare. Six of the MAC serovars have not yet been definitively classified. MAC infections currently account for approximately 50% of the pathogenic isolates identified by mycobacteriology labs and are most common among AIDS and other immunocompromised patients. Early diagnosis and treatment of MAC infections can improve and prolong the lives of infected individuals.
The diagnosis of mycobacterial infections has traditionally been dependent on acid-fast staining and cultivation of the organism, followed by biochemical assays. These procedures are time-consuming, and a typical diagnosis using conventional culture methods can take as long as six weeks. Automated culturing systems such as the BACTEC.TM. system (Becton Dickinson Microbiology Systems, Sparks, Md.) can decrease the time for diagnosis to one to two weeks. However, there is still a need to reduce the time required for diagnosing Mycobacterial infections to less than a week, preferably to one day or less. Nucleic acid amplification is a powerful technology which allows rapid detection of specific target sequences. It is therefore a promising technology for rapid detection and identification of Mycobacteria. Examples of nucleic acid amplification technologies known in the art are Polymerase Chain Reaction (PCR: U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), Strand Displacement Amplification (SDA--G. Walker, et al. 1992. Proc. Nat. Acad. Sci. U.S.A. 89, 392-396; G. Walker, et al. 1992. Nucl. Acids Res. 20, 1691-1696; U.S. Pat. No. 5,270,184; U.S. Pat. No. 5,455,166; published European Patent Application No. 0 684 315), nucleic acid sequence based amplification (NASBA: U.S. Pat. No. 5,130,238 to Cangene), transcription based amplification (TAS--D. Kwoh, et al. 1989. Proc. Nat. Acad Sci. U.S.A. 86, 1173-1177), self-sustained sequence replication (3SR: J. Guatelli, et al. 1990. Proc. Nat. Acad Sci. U.S.A. 87, 1874-1878) and the Q.beta. replicase system (P. Lizardi, et al. 1988. BioTechnology 6, 1197-1202).
Isothermal amplification methods such as SDA and 3 SR have particular advantages in diagnostics, as they do not require the high/low temperature cycling characteristic of methods such as the PCR. They are therefore simpler protocols and require less specialized equipment to perform. However, isothermal amplification methods such as SDA generally are not capable of amplifying targets as large as those amplifiable by PCR. Small target sequences severely restrict the ability to design primers and probes with the desired specificity for detection of a given target because the proximity of appropriate amplification primer binding sites becomes a factor and there is less sequence available in the amplification product for assay probe design.
Initially, SDA was developed for use at temperatures between about 35.degree. C. and 45.degree. C. ("conventional SDA"). Recently, it has been adapted to higher temperatures using thermophilic polymerases and restriction endonucleases ("thermophilic SDA" or "tSDA") as described in published European Patent Application No. 0 684 315. The tSDA system provides the advantages of increased speed and specificity as compared to conventional SDA. While the target binding sequences of amplification primers designed for use in conventional SDA generally will function in tSDA, they are usually shorter and amplification efficiency may therefore be reduced at the higher temperatures of tSDA. Also, as is the case for primer design in conventional SDA, apparently minor modifications in the target binding sequence of primers for tSDA (such as lengthening it) often have unpredictable effects on amplification efficiency. In contrast, primers comprising the target binding sequences of primers designed for tSDA usually function efficiently when adapted to amplification primers for conventional SDA or other amplification reactions.
The heat shock proteins are a family of proteins which are expressed in elevated amounts when an organism is challenged by an increase in temperature. The heat shock proteins are highly conserved (R. J. Garcia, et al. 1989. Infection and Immunity 57, 204-212; R. S. Gupta, et al. 1992. J. Bacteriology 174, 4594-4605). The dnaJ gene codes for a 42 kd heat-shock protein believed to be involved in the cellular stress response. M. tuberculosis was the first of the mycobacteria for which the nucleotide sequence of the dnaJ gene was determined (R. B. Lathigra, et al. 1988. Nucl. Acids Res. 16, 1636). The nucleotide sequence of a segment of the dnaJ gene of M. leprae was subsequently determined (S. S. Harvey, et al. 1993. J. Gen. Microbial. 139, 2003-2008). Later, using the M. tuberculosis sequence published by R. B. Lathigra et al., supra, S. I. Takewaki, et al. (1993. J. Clin. Microbiol. 31, 446-450) developed a set of genus-specific PCR primers which amplify a 236-bp fragment of the dnaJ gene (bp 1394-1629) from a broad range of mycobacterial species, including M. avium and M. intracellulare. Species-specific oligonucleotide probes which allowed identification of M. tuberculosis, M. avium, M. intracellulare, and M. kansasii following genus-specific amplification by PCR were also reported. The dnaJ gene of nineteen species of mycobacteria was then sequenced and used to determine phylogenetic relationships and to differentiate species on the basis of species-specific restriction sites in the gene (S. I. Takewaki, et al. 1994. Int. J. Syst. Bacteriol. 44, 159-166). Japanese Kokai Patent No. 6-133775 (Takewaki, et al., published May 17, 1994) discloses a genus-specific amplification primer pair for PCR and several species-specific probes derived from the dnaJ gene of mycobacteria.
Certain terms used herein are defined as follows:
An amplification primer is a primer for amplification of a target sequence by extension of the primer after hybridization to the target sequence. The 3' end of an SDA amplification primer (the target binding sequence) hybridizes at the 3' end of the target sequence. The target binding sequence confers target specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease near its 5' end. The recognition site is for a restriction endonuclease which will nick one strand of a DNA duplex when the recognition site is hemimodified, as described by G. Walker, et al. (1992. PNAS, supra). The SDA amplification primer may also be referred to as the "S" primer (e.g., S.sub.1 and S.sub.2 when a pair of amplification primers is used for amplification of a double stranded sequence). For amplification methods which do not require specialized sequences at the ends of the target, the amplification primer generally consists essentially of only the target binding sequence. For example, amplification of a target sequence according to the invention using the PCR will employ amplification primers consisting of the target binding sequences of the amplification primers in Table 1. For amplification methods which require specialized sequences other than a restriction endonuclease recognition site appended to the target (e.g., an RNA polymerase promoter for 3SR, NASBA or transcription based amplification), the required specialized sequence may be linked to the target binding sequence shown in Table 1 using routine methods such as chemical synthesis for preparation of the oligonucleotides.
A bumper primer or external primer is a primer used to generate targets which can be amplified by SDA. The bumper primer anneals to a target sequence upstream of the amplification primer such that extension of the bumper primer displaces the downstream amplification primer and its extension product. Bumper primers may also be referred to as "B" primers (e.g., B.sub.1 and B.sub.2 when a pair of bumper primers is used to displace the extension products of a pair of amplification primers). Extension of bumper primers is one method for displacing the extension products of amplification primers, but heating is also suitable in certain amplification reactions.
The terms target or target sequence refer to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, the complementary second strand of the original nucleic acid sequence to be amplified, and either strand of a copy of the original sequence which is produced by the amplification reaction. These copies also serve as amplifiable target sequences by virtue of the fact that they comprise copies of the original target sequences to which the amplification primers hybridize.
Copies of the target sequence which are generated during the amplification reaction are referred to as amplification products, amplimers or amplicons.
The term extension product refers to the single-stranded copy of a target sequence produced by hybridization of an amplification primer and extension of the amplification primer by polymerase using the target sequence as a template.
The term assay probe refers to any of the oligonucleotides used in the detection or identification portion of an assay. In the present invention, the assay probes are probes used for complex-, group- or species-specific detection or identification of Mycobacteria. Detector probes and capture probes are examples of assay probes.
The assay region or assay region sequence is the portion of a target sequence, or other nucleic acid, to which an assay probe hybridizes.
The term species-specific refers to detection or amplification in a species of organism without substantial detection or amplification in other species of the same genus or species of a different genus. Genus-specific refers to detection or amplification in the majority of the species of a genus, without substantial detection or amplification in the species of a different genus. Group- or complex-specific refers to detection or amplification in a majority of related species in a selected group (e.g., MAC) without substantial detection or amplification in other species of the same genus or species of a different genus.